This invention relates to materials and methods for the detection of DNA 5-cytosine methyltransferase (5-C-DNA methyltransferase) and more particularly relates to a novel oligodeoxyribonucleotide, i.e. ODN, as a substrate for selective detection and quantification of mammalian 5-C-DNA methyltransferase.
Regulation of gene expression in eukaryotic organisms involves many different biological mechanisms that operate independently of one another. DNA methylation is among these mechanisms associated with the regulation of gene expression. The enzyme which is responsible for this process, DNA methyltransferase, acts by transferring a methyl group from S-adenosylmethionine to a cytosine residue in a CG-sequence of DNA to give an altered sequence containing 5-methylcytosine. Not all CG sequences in DNA are methylated by the enzyme and not all DNA methyl transfer reactions are involved in gene expression, but there is a consistent, well-documented correlation between DNA methylation and the inhibition of the activity of many genes.
Attempts to clarify the role that DNA methylation plays in gene expression have been hampered by the lack of a specific inhibitor of DNA methyltransferase. In this regard, the compounds 5-azacytidine (azaC) and 5-aza-2'-deoxycytidine (azadC) have been used to study inhibition of DNA methylation. Both azaC and azadC are metabolized and are ultimately incorporated into cellular DNA. Subsequently, DNA which contains azadC directly inhibits DNA methyltransferase and thus, the overall process of DNA methylation. However, DNA which contains azadC also interferes with a variety of other DNA protein-interactions and so the cellular effects of azadC can not be ascribed solely to its ability to inhibit DNA methylation. There remains a need for the systematic design and synthesis of totally specific inhibitors of DNA methyltransferase.
For example, methylation of Cytosine (C) residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of at least some human cancers. In vertebrates, it appears that the same DNA methyltransferase (DNA MTase) maintains pre-existing patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions.
In vertebrate cells, about 3% of cytosine (C) residues in DNA have a methyl group on carbon 5 and 5-methyl cytosine (5 mC) is the only naturally occurring modified base so far detected in DNA. Enzymatic methylation of Cytosine residues in DNA occurs postreplicatively and primarily involves C residues in CpG dinucleotides, although methylation has been observed at C residues 5' of other nucleotides. The extent and pattern of methylation of genomic DNA is species- and tissue-specific, which implies that the pattern of methylation is faithfully inherited in all cells of common lineage within a tissue. Analysis of methylation patterns of specific genes during development suggests that patterns established in sperm and oocytes are lost during early development, that regions other than CpG islands become almost fully methylated, and that loss of methylation occurs at specific sites in tissues where a gene is expressed.
Although not all genes are regulated by methylation, hypomethylation at specific sites or in specific regions in a number of genes is correlated with active transcription. DNA methylation in vitro can prevent efficient transcription of genes in cell-free systems or transient expression of transfected genes; methylation of C residues in some specific cis-regulatory regions can also block or enhance binding of transcription factors or repressors. DNA methylation is involved in inactivation of one of the two X chromosomes in female mammalian somatic cells, and allele-specific methylation has been proposed as a factor in genomic imprinting. The most direct evidence for the importance of DNA methylation in development is the demonstration that homozygous mutation in murine DNA 5-cytosine methyltransferase (5-C-MTASE) leads to impaired embryonic development.
Conversely, disruption of normal patterns of DNA methylation has been linked to development of cancer. The 5-methylcytidine (5MeC) content of DNA from tumors and tumor-derived cell lines is generally lower than in normal tissues, although increased methylation of CpG sites occurs in some genes and chromosome regions. While these observations support the concept that methylation patterns are established in the embryo and altered during carcinogenesis by a combination of de novo methylation and loss of methylation in a time-, sequence-, and tissue-specific manner, the mechanism(s) by which these changes occur and are regulated with such apparent precision has not been defined.
The processes involved in regulating de novo methylation are particularly puzzling. As would be predicted for an enzyme that maintains established patterns of methylation during DNA replication, mammalian DNA MTases have a much greater capacity for methylating hemimethylated CpG sites in double-stranded (ds) DNA than completely unmethylated sites. However, since the gene encoding mammalian DNA 5-C-MTase is present as a single copy per haploid genome and there is no direct evidence for the existence of a separate de novo DNA MTase, it appears that the same enzyme must carry out both functions.
It should be pointed out that bacterial 5-C-DNA methyltransferases do not function in the same way as and are not the same as mammalian 5-C-DNA methyltransferases but do have sufficient similarity in mechanism that to date the presence of bacterial 5-C-DNA methyltransferase can interfere with detection methods for mammalian 5-C-DNA methyltransferase.
In any case, mammalian DNA methyltransferases are involved in gene expression and the activity of this enzyme is elevated in the colon mucosa of patients at risk for colon cancer. It has also been shown that lowering the level and activity of DNA 5-MTase also lowers the incidence of colon cancer in transgenic mice that develop this disease spontaneously. It is therefore important to be able to detect and quantify mammalian 5-C-DNA methyltransferases and distinguish them from other 5-C-DNA methyltransferases which cause different functional effects.